New crystal form of ergothioneine and preparation method thereof

Abstract

The invention provides a novel crystal form of ergothioneine and a preparation method thereof, and belongs to the technical field of medical crystallization. The method for preparing the new crystal form of ergothioneine provided by the invention is simple to operate, low in energy consumption and high in economical efficiency, the product has good chemical stability, the yield is more than 90%, the purity reaches more than 99.90%, the preparation process is stable, the reproducibility is high, and the method has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical crystallization, and particularly relates to a new crystal form of ergothioneine and a preparation method thereof. Background Art

[0002] L-Ergothioneine (EGT), chemical formula is C9H 15 N3O2S, with a relative molecular mass of 229.3, has a chemical name of 2-mercaptohistidine trimethyl salt, and a CAS number of 497-30-3. It exists in two isomers, namely thiol and thione, and its structural formula is as follows:

[0003]

[0004] At room temperature, EGT appears as a white crystalline powder and is easily soluble in water, but has low solubility in most organic solvents.

[0005] Ergothioneine is a natural rare chiral amino acid strong antioxidant and an important physiologically active substance in the body. It not only has the functions of anti-oxidation, scavenging free radicals, detoxification, and maintaining DNA biosynthesis, but also has the functions of preventing ultraviolet radiation damage, regulating redox reactions in cells, participating in intracellular energy regulation, and acting as a cell physiological protector. Due to the biological and pharmacological properties of ergothioneine and its advantages of safety and stability, it has shown good application prospects in the fields of cosmetics, functional foods, pharmaceuticals, therapeutics, and biomedicine. It is a new type of fermented product with high added value and has become a hot topic of research.

[0006] Polymorph refers to the crystallization of the same solid substance under different physical environment conditions, forming a crystal with significant differences in internal structure, and polymorph is a common phenomenon of drug molecules, and different crystal forms can show different physical and chemical properties, thereby affecting the efficacy of medicine. At present, EGT has been publicly reported to have two kinds of crystal forms, namely EGT anhydrous crystal form and EGT dihydrate, which are respectively named as crystal forms A and B. Patent application documents (CN118221592A, CN118908894A) disclose the preparation methods of these two kinds of thioneine crystals respectively, and the yields of the preparation methods are respectively 92.5% and 84%. Commercially available EGT is crystal form A. However, commercially available EGT has problems such as easy moisture absorption and poor stability. Therefore, it is of great significance to develop a method for preparing a new crystal form of thioneine with stable performance. Summary of the invention

[0007] In view of the problems existing in the prior art, the object of the present invention is to provide a new crystal form of ergothioneine and a preparation method thereof.

[0008] The invention provides a thioneine crystal form. In the X-ray powder diffraction of the crystal form, 2θ diffraction angles have characteristic absorption peaks at 5.8±0.2°, 6.9±0.2°, 11.6±0.2°, 13.9±0.2°, 19.7±0.2°, 21.0±0.2°, 22.7±0.2°, 24.8±0.2°, 25.9±0.2°, 28.3±0.2° and 29.5±0.2°.

[0009] Furthermore, in the X-ray powder diffraction of the crystal form, the peak area percentage % of the characteristic peak at 2θ diffraction angle is:

[0010] Diffraction angle 2θ Peak area percentage % 5.8±0.2° 72.73％ 6.9±0.2° 80.15％ 11.6±0.2° 29.11％ 13.9±0.2° 44.47％ 19.7±0.2° 11.20％ 21.0±0.2° 41.18％ 22.7±0.2° 100.00％ 24.8±0.2° 50.88％ 25.9±0.2° 36.66％ 28.3±0.2° 14.06％ 29.5±0.2° 55.56％ .

[0011] Furthermore, the X-ray powder diffraction spectrum of the crystal form is as follows Figure 1 shown.

[0012] Further, the DSC of the crystalline form is as follows Figure 2 As shown, a melting peak appears at 265±1°C.

[0013] The present invention also provides a method for preparing the above-mentioned ergothioneine crystal form, the method comprising the following steps:

[0014] (1) prepare the aqueous solution of thioneine;

[0015] (2) adding an organic solvent into the aqueous solution of the thioneine of step (1), stirring and eluting, crystallization;

[0016] (3) crystal growth;

[0017] (4) filtering and drying to obtain thioneine crystal form.

[0018] Further, in step (1), the temperature of the water is 10 to 80° C., preferably 20° C.; the concentration of ergothioneine in the aqueous solution of ergothioneine is 0.5 to 3.0 mol / L, preferably 0.8 mol / L.

[0019] Furthermore, in step (2), the volume ratio of water to organic solvent is 5:0.5-5, preferably 5:0.8-4.5; the stirring conditions are: stirring at a feeding rate of 100-500 μL / min, at a temperature of 0-40°C, and at a stirring rate of 100-600 r / min, preferably: stirring at a feeding rate of 300 μL / min, at a temperature of 30°C, and at a stirring rate of 400 r / min; the organic solvent is one or a mixture of two or more of methanol, ethanol, isopropanol, acetone, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, and ethylene glycol, preferably dimethyl sulfoxide.

[0020] Furthermore, in step (3), the crystal growing time is 0.5 to 1.5 hours, preferably 0.5 hours.

[0021] Furthermore, in step (4), the drying temperature is 30 to 60° C., preferably 50° C.; and the drying time is 2 to 10 hours, preferably 6 hours.

[0022] The invention also provides a pharmaceutical composition, which is a preparation prepared by taking the ergothioneine crystal form as an active ingredient and adding auxiliary materials commonly used in pharmacy.

[0023] The present invention also provides use of the ergothioneine crystal form in preparing antioxidant cosmetics, antioxidant health foods or antioxidant medicines.

[0024] The present invention prepares a new crystal of thioneine, and compared with the thioneine crystal prepared by patent application document (CN118221592A), the thioneine new crystal prepared by the present invention still has 99.93% stability in 30 days; and the thioneine crystal prepared by patent application document (CN118221592A) has a maximum retention rate of 99% after illumination four weeks. Therefore, the thioneine new crystal prepared by the present invention has better chemical stability.

[0025] The present invention also provides a method for preparing the above-mentioned thioneine new crystal form, the preparation method is simple to operate, less energy consumption, high economy, and the preparation process is stable and highly reproducible. The preparation method can obtain a yield of more than 90%, and the purity reaches more than 99.90% of the thioneine new crystal form. The present invention further optimizes the process conditions of the reaction, and the preferred process conditions shown in Example 1 are obtained by screening. The highest yield of the thioneine new crystal form prepared by the present invention can reach 94%, compared with the yield (92.5%, 84%) of preparing thioneine crystals in patent application documents (CN118221592A, CN118908894A), the yield of the thioneine new crystal form obtained by the preparation method of the present invention is higher.

[0026] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0027] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1X-ray powder diffraction pattern of the new crystal form of EGT in Example 1 of the present invention.

[0029] Figure 2 DSC-TG analysis chart of the new crystal form of EGT in Example 1 of the present invention.

[0030] Figure 3 FT-IR image of the new crystal form of EGT in Example 1 of the present invention. DETAILED DESCRIPTION

[0031] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0032] Embodiment 1, the preparation of the new crystal form of thioneine

[0033] At 40°C, 0.9172 g of ergothioneine (EGT) solid was dissolved in 5 mL of deionized water to form a clear solution; stirring was started and the stirring rate was controlled at 400 r / min; the temperature in the crystallizer was controlled at 30°C, and 1.2 mL of dimethyl sulfoxide was added dropwise to the clear EGT solution at a feeding rate of 300 μL / min; the stirring rate was maintained and the crystal was grown for 0.5 h; the product was filtered and a filter cake was obtained by vacuum filtration; the filter cake was placed in an oven and dried at a constant temperature of 50°C for 6 h; after drying, the new EGT crystal was obtained.

[0034] The X-ray powder diffraction pattern of the product has diffraction peaks at diffraction angles 2θ=5.8±0.2°, 6.9±0.2°, 11.6±0.2°, 13.9±0.2°, 19.7±0.2°, 21.0±0.2°, 22.7±0.2°, 24.8±0.2°, 25.9±0.2°, 28.3±0.2°, and 29.5±0.2°. DSC-TG results show that a melting peak occurs at 265.7°C. The product has a white powder appearance, a product purity of 99.96%, and a process yield of 94%.

[0035] Embodiment 2, the preparation of the new crystal form of thioneine

[0036] At 40°C, 1.0318 g of EGT solid was dissolved in 5 mL of deionized water to form a clear solution; stirring was started and the stirring rate was controlled at 300 r / min; the temperature in the crystallizer was controlled at 40°C, and 2.0 mL of acetone was added dropwise to the clear EGT solution at a feeding rate of 400 μL / min; the stirring rate was maintained and the crystal was grown for 1.0 h; the product was filtered and a filter cake was obtained by vacuum filtration; the filter cake was placed in an oven and dried at a constant temperature of 60°C for 2 h; after drying, the new EGT crystal form was obtained.

[0037] The X-ray powder diffraction pattern of the product has diffraction peaks at diffraction angles 2θ=5.8±0.2°, 6.9±0.2°, 11.6±0.2°, 13.9±0.2°, 19.7±0.2°, 21.0±0.2°, 22.7±0.2°, 24.8±0.2°, 25.9±0.2°, 28.3±0.2°, and 29.5±0.2°. DSC-TG results show that a melting peak occurs at 265.7°C. The product has a white powder appearance, a product purity of 99.93%, and a process yield of 93%.

[0038] Embodiment 3, the preparation of the new crystal form of thioneine

[0039] At 30°C, 0.8026 g of EGT solid was dissolved in 5 mL of deionized water to form a clear solution; stirring was started and the stirring rate was controlled at 200 r / min; the temperature in the crystallizer was controlled at 25°C, and 0.8 mL of ethylene glycol was added dropwise to the clear EGT solution at a feeding rate of 100 μL / min; the stirring rate was maintained and the crystal was grown for 0.5 h; the product was filtered and a filter cake was obtained by vacuum filtration; the filter cake was placed in an oven and dried at a constant temperature of 30°C for 8 h; after drying, the new EGT crystal form was obtained.

[0040] The X-ray powder diffraction pattern of the product has diffraction peaks at diffraction angles 2θ=5.8±0.2°, 6.9±0.2°, 11.6±0.2°, 13.9±0.2°, 19.7±0.2°, 21.0±0.2°, 22.7±0.2°, 24.8±0.2°, 25.9±0.2°, 28.3±0.2°, and 29.5±0.2°. DSC-TG results show that a melting peak occurs at 265.7°C. The product has a white powder appearance, a product purity of 99.95%, and a process yield of 92%.

[0041] Embodiment 4, the preparation of the new crystal form of thioneine

[0042] At 20°C, 0.6879 g of EGT solid was dissolved in 5 mL of deionized water to form a clear solution; stirring was started and the stirring rate was controlled at 100 r / min; the temperature in the crystallizer was controlled at 30°C, and 2.0 mL of acetonitrile was added dropwise to the clear EGT solution at a feeding rate of 200 μL / min; the stirring rate was maintained and the crystal was grown for 1.5 hours; the product was filtered and a filter cake was obtained by vacuum filtration; the filter cake was placed in an oven and dried at a constant temperature of 50°C for 10 hours; after drying, the new EGT crystal form was obtained.

[0043] The X-ray powder diffraction pattern of the product has diffraction peaks at diffraction angles 2θ=5.8±0.2°, 6.9±0.2°, 11.6±0.2°, 13.9±0.2°, 19.7±0.2°, 21.0±0.2°, 22.7±0.2°, 24.8±0.2°, 25.9±0.2°, 28.3±0.2°, and 29.5±0.2°. DSC-TG results show that a melting peak occurs at 265.7°C. The product has a white powder appearance, a product purity of 99.92%, and a process yield of 91%.

[0044] Embodiment 5, the preparation of the new crystal form of thioneine

[0045] At 25°C, 0.8026 g of EGT solid was dissolved in 5 mL of deionized water to form a clear solution; stirring was started and the stirring rate was controlled at 500 r / min; the temperature in the crystallizer was controlled at 20°C, and 3.0 mL of a methanol-ethanol mixed solution was added dropwise to the clear EGT solution at a feeding rate of 300 μL / min; the stirring rate was maintained and the crystal was grown for 1.0 h; the product was filtered and a filter cake was obtained by vacuum filtration; the filter cake was placed in an oven and dried at a constant temperature of 40°C for 7 h; after drying, the new EGT crystal form was obtained.

[0046] The X-ray powder diffraction pattern of the product has diffraction peaks at diffraction angles 2θ=5.8±0.2°, 6.9±0.2°, 11.6±0.2°, 13.9±0.2°, 19.7±0.2°, 21.0±0.2°, 22.7±0.2°, 24.8±0.2°, 25.9±0.2°, 28.3±0.2°, and 29.5±0.2°. DSC-TG results show that a melting peak occurs at 265.7°C. The product has a white powder appearance, a product purity of 99.91%, and a process yield of 93%.

[0047] Embodiment 6, the preparation of the new crystal form of thioneine

[0048] At 25°C, 0.8026 g of EGT solid was dissolved in 5 mL of deionized water to form a clear solution; stirring was started and the stirring rate was controlled at 300 r / min; the temperature in the crystallizer was controlled at 15°C, and 4.5 mL of N,N-dimethylformamide was added dropwise to the clear EGT solution at a feeding rate of 500 μL / min; the stirring rate was maintained and the crystal was grown for 1.5 hours; the product was filtered and a filter cake was obtained by vacuum filtration; the filter cake was placed in an oven and dried at a constant temperature of 30°C for 10 hours; after drying, the new EGT crystal form was obtained.

[0049] The X-ray powder diffraction pattern of the product has diffraction peaks at diffraction angles 2θ=5.8±0.2°, 6.9±0.2°, 11.6±0.2°, 13.9±0.2°, 19.7±0.2°, 21.0±0.2°, 22.7±0.2°, 24.8±0.2°, 25.9±0.2°, 28.3±0.2°, and 29.5±0.2°. DSC-TG results show that a melting peak occurs at 265.7°C. The product has a white powder appearance, a product purity of 99.94%, and a process yield of 93%.

[0050] The results of Examples 1 to 6 show that the purity and yield of the new thioneine crystal obtained under the conditions of Example 1 are the highest, with a purity of 99.96% and a yield of 94%.

[0051] The new EGT crystal form obtained in Examples 1 to 6 is an anhydrous crystal form, and the chemical formula is C9H 15 N3O2S.

[0052] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0053] Experimental Example 1: Testing the chemical properties of the new crystalline form of ergothioneine

[0054] The chemical properties of the new crystal form of EGT obtained in Examples 1-6, i.e., the new crystal form of EGT described in the present invention, were tested.

[0055] 1) The chemical stability of the new EGT crystal prepared by the present invention was investigated. During storage for 30 days at 30°C ± 5°C and RH 40 ± 5%, the color, purity and morphology of the product did not change, indicating that the crystal had good chemical stability. The investigation results are shown in Table 1.

[0056] Table 1 Chemical stability study of the new EGT crystal form described in the present invention

[0057] 5 days 10 days 15 days 20 days 25 days 30 days Characteristics White crystal White crystal White crystal White crystal White crystal White crystal Example 1 Purity 99.96％ 99.95％ 99.95％ 99.94％ 99.93％ 99.93％ Example 2 Purity 99.93％ 99.93％ 99.93％ 99.92％ 99.92％ 99.92％ Example 3 Purity 99.95％ 99.95％ 99.94％ 99.94％ 99.94％ 99.93％ Example 4 Purity 99.92％ 99.92％ 99.91％ 99.91％ 99.90％ 99.90％ Example 5 Purity 99.91％ 99.91％ 99.91％ 99.90％ 99.90％ 99.90％ Example 6 Purity 99.94％ 99.94％ 99.94％ 99.93％ 99.93％ 99.92％

[0058] 2) If Figure 1 The X-ray powder diffraction pattern of the new EGT crystal obtained in Example 1 and the X-ray powder diffraction pattern of the new EGT crystal obtained in Examples 1-6 are shown. Figure 1 To.

[0059] 3) The EGT new crystal form obtained by the present invention is subjected to DSC-TG analysis. The specific method of DSC-TG analysis is a conventional method in the prior art. The new crystal form obtained in Example 1 is tested to obtain a DSC-TG analysis diagram of the EGT new crystal form as shown in FIG. Figure 2 As shown, its DSC analysis chart shows that there is a melting peak at 265° C. From the information in the figure, it can be determined that the new EGT crystal obtained in the embodiment has excellent thermal stability.

[0060] 4) Figure 3 This is the FT-IR image of the new EGT crystal form obtained in Example 1. The FT-IR results of Examples 1-6 are consistent.

[0061] In summary, the present invention provides a new crystal form of thioneine and a preparation method thereof. The operation of preparing the new crystal form of thioneine provided by the present invention is simple, energy-saving, and economical, and the product has good chemical stability, a yield of more than 90%, a purity of more than 99.90%, and a stable preparation process, high reproducibility, and good application prospects.

Claims

1. A thioneine crystal form, characterized in that, In the X-ray powder diffraction of the crystal form, the 2θ diffraction angles have characteristic absorption peaks at 5.8±0.2°, 6.9±0.2°, 11.6±0.2°, 13.9±0.2°, 19.7±0.2°, 21.0±0.2°, 22.7±0.2°, 24.8±0.2°, 25.9±0.2°, 28.3±0.2°, and 29.5±0.2°.

2. the thioneine crystal form according to claim 1, is characterized in that, In the X-ray powder diffraction of the crystal form, the peak area percentage % of the characteristic peak at 2θ diffraction angle is: 。 3. the thioneine crystal form according to claim 1, is characterized in that, The X-ray powder diffraction spectrum of the crystal form is shown in FIG1 .

4. the thioneine crystal form according to claim 1, is characterized in that, The DSC of the crystalline form is shown in FIG2 , which shows a melting peak at 265±1° C.

5. A method for preparing the thioneine crystal form described in any one of claims 1-4, characterized in that, The method comprises the following steps: (1) prepare the aqueous solution of thioneine; (2) adding an organic solvent into the aqueous solution of the thioneine of step (1), stirring and eluting, crystallization; (3) crystal growth; (4) filtering and drying to obtain thioneine crystal form.

6. The method according to claim 5, characterized in that In step (1), the temperature of the water is 10-80° C., preferably 20° C.; the concentration of ergothioneine in the aqueous solution of ergothioneine is 0.5-3.0 mol / L, preferably 0.8 mol / L.

7. The method according to claim 5, characterized in that In step (2), the volume ratio of water to organic solvent is 5:0.5-5, preferably 5:0.8-4.5; the stirring conditions are: stirring at a feeding rate of 100-500 μL / min, at a temperature of 0-40°C, and at a stirring rate of 100-600 r / min, preferably: stirring at a feeding rate of 300 μL / min, at a temperature of 30°C, and at a stirring rate of 400 r / min; the organic solvent is one or a mixture of two or more of methanol, ethanol, isopropanol, acetone, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, and ethylene glycol, preferably dimethyl sulfoxide.

8. The method according to claim 5, characterized in that In step (3), the crystal growing time is 0.5 to 1.5 hours, preferably 0.5 hours.

9. A pharmaceutical composition, characterized in that The invention discloses a preparation which is prepared by taking the ergothioneine crystal form described in any one of claims 1 to 4 as an active ingredient and adding auxiliary materials commonly used in pharmacy.

10. the purposes of the thioneine crystal form described in any one of claims 1-4 in preparing antioxidant cosmetics, antioxidant health food or antioxidant medicine.

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